Figure 2.

Construction of four DH10B-based hosts carrying a tightly regulated trfA gene that supplies, but only upon induction, the TrfA replication protein. (A) A representation of an integration plasmid and of a fragment of the host genome with the attB site for site-specific recombination. Four integration plasmids carrying four various trfA copy-up mutations have been constructed (see Table 1), as described in Methods. Each integration plasmid carries a cassette consisting of araC–P_BAD fused to the specific trfA gene copy-up mutant. All integration plasmids have (1) an easily removable NotI-flanked ori of plasmid pBR322, and (2) the attP_λ site for site-specific integration into attB of the DH10B host genome, as shown below the plasmid drawing. (B) A diagram of the genomic segment of the host upon recombination of the trfA-integration plasmid. Such hosts permit conditional, tightly regulated synthesis of the TrfA protein. Experimental details on Int-mediated integration of the four plasmids into the DH10B host strains (Table 2) are described in Methods. TT1 represents the t1 and t2 terminators (both clockwise) from rrnB; TT2 represents the t_L3 (clockwise) and t_L1 (anticlockwise) terminators of phage λ.